The present invention relates to analytical chemistry and, more particularly, to correlated multidimensional chromatography. A major objective of the present invention is to provide for improved correlation of peaks from independent chromatographic separations of sample components.
Analytical chemistry has provided for fundamental advances in environmental and medical sciences. At the heart of analytical chemistry are techniques that provide for the separation, identification and quantification of components of a sample. "Chromatography" denotes a class of analytic methods that separate a sample into components as it moves along a separation path and typically provide for sequential quantification of the separated components. Herein, "chromatography" includes not only gas chromatography (GC) and liquid chromatography (LC), but also capillary electrophoresis (CE) and gel capillary electrophoresis (GCE).
In GC and LC, a sample is carried by a fluid (gas or liquid) that flows past a stationary support. As they flow past the support, some sample molecules are adsorbed to the support. The adsorption is reversible in that adsorbed sample molecules are desorbed to the fluid. Adsorbed sample molecules are considered to be in a "stationary phase", while desorbed molecules are considered to be in a "mobile phase".
As the number of adsorbed molecules increases, the rate of desorption increases until adsorption and desorption equalize. When equality is achieved, the proportions of each sample component in the mobile and stationary phases are in equilibrium. The partitioning of a component between the stationary phase and the mobile phase at equilibrium is a function of the component, the support material and the carrier fluid. For a given support material and carrier fluid, partitioning is a function of the sample components.
Sample components with a relatively large proportion of their molecules in the stationary phase, spend relatively less time in the mobile phase. Therefore, the migration rates for such components are less than the migration rates for sample components with relatively small proportions of their molecules in the stationary phase. It is this difference in migration rates that effects liquid and gas chromatographic separations.
In CE, an electric field is applied along a separation path. The electric field propels molecules in proportion to their charge, while viscous drag counters the propulsion in relation to the molecules size. Thus, CE separates sample components according to their charge-to-mobility ratios.
A detector, for example a thermal conductivity detector (for GC) or an ultraviolet absorption detector (for LC or CE), provides a time-varying output as separated sample components exit the solid phase. This time-varying output corresponds to the spatial distribution of the concentrations of the separated sample components. The time varying output can be recorded, providing a spatial representation of the spatial distribution of concentrations. The recorded output is referred to as a chromatogram. A typical chromatogram has a series of peaks. Each peak corresponds to a component band, or a group of unresolved component bands. Areas under peaks can be used to quantify the components.
One of the challenges of chromatography is to identify the sample component associated with a peak. Under carefully controlled conditions, sample components can be identified by the "retention time" at which they are detected. Test runs can be used to construct a table of retention times for expected analytes separated under specified conditions. One can then work backward from the retention time of a peak to determine the associated component. This identification approach is limited by the fact that many chemical moieties can have similar retention times. Furthermore, slight variations in conditions can cause retention times to vary. Providing tolerances for this variation greatly expands the number of chemical moieties that could correspond to a given retention time.
Identification can proceed much more surely by isolating the separated components and applying some well established identification technique. For example, GC/MS systems (gas chromatography/mass spectroscopy) systems separate components using gas chromatography and identify them using mass spectroscopy. Alternative identification techniques involve capturing eluting bands in separate vials. Various spectrographic and chemical tests can be applied to identify the component associated with the separated band. However, such physical isolation of separated components can be cumbersome when hundreds of peaks are involved.
In a "hyphenated" system, the step of collecting separate components can be avoided by appending a "fast" column to the end of a "slow" column. For example, a "fast" CE column can be abutted to the end of a "slow" LC column. The effluent of the slow column is sampled; the resulting sample slices are then run through,the fast column before the next slice enters the fast column. A single detector at the end of the fast column in effect identifies: 1) a "gross retention time" associated with the slice in which 1.5 a peak is detected, and thus the retention time in the slow column; and 2) a "fine retention time" the peak spent in the fast column. This permits two retention times to be associated with each band. Assuming the selectivity characteristics of the fast and slow column are different, the second retention time can be used to separate some chemical species that could not be separated on the basis of the first retention time alone. Thus, the chances of a unique identification are increased.
In practice, the slow and fast columns must be operated non-optimally in order to work well together. The resolutions of each are impaired by the need to infrequently sample from the slow column and drastically speed up the elution rate for the fast column. The lower resolutions broaden the peaks, causing severe overlaps and reducing the precision of identifications. Furthermore, sensitivities are limited because of the small sample sizes required for the fast column, which, of necessity, is usually small.
Correlated two-dimensional chromatography can enhance the identification capabilities of chromatography without requiring further analysis of separated components. In correlated two-dimensional chromatography, two columns of different selectivities are used. Two allotments of a sample are run respectively through the two columns. The runs are designed to be independent so that conditions can be optimized for each column. In practice, the runs can be concurrent to minimize analysis time. Generally, substances that are unresolved in one column can be resolved in the other. The results of each chromatogram can be used to confirm determinations based on the other chromatogram.
Correlated multi-dimensional chromatography is the generalization of this approach to two or more columns with different selectivities. Since runs are performed independently, each run can be optimized individually for resolution and speed. This provides a major advantage over the hyphenated approach in which resolution in each column is compromised so that fast column can separate the components in each sample slice within one sampling duration. Thus, a major advantage of this approach over the hyphenated approach is that both runs can be individually optimized for high sensitivity and high speed.
The challenge of correlated multidimensional chromatography is "correlating" peaks in two or more chromatograms. "Correlating" involves assigning a correspondence among peaks presumed to represent the same sample component. It can be difficult to determine which peak of one chromatogram corresponds with a given peak of the another chromatogram. Once the correct assignment is made, one can work backwards from tables of retention times for both runs to identify the component. To the extent that the selectivities for the runs are independent, the information on common-component peaks is generally much less ambiguous than the information from a single peak.
Unfortunately, it can be very difficult to determine which peaks are to be commonly assigned. For example, if there are 100 peaks in each of two chromatograms, there are 100! possible sets of peak pairings, assuming that the same 100 components are represented in each chromatogram. The number of possible correlations can be larger when overlapping is considered. Correlation techniques typically involve methods for excluding possible correlations, with an objective of being left with a single correlation. Many of "incorrect" correlations can be eliminated by comparing areas under peaks. This approach is limited by the usual presence of overlapping component bands that generate convolved peaks. Available knowledge about the sample can help exclude some possible correlations by the technique of training the particular apparatus on components expected to be present. However, this technique is not of use in correlating peaks for components not previously trained on. In some cases, however, there may be no basis for selecting among hundreds of possible correlations.
What is needed is a correlated multi-dimensional chromatographic method that provides for both high-resolution multi-dimensional separations and high-confidence co-assignment of peaks without requiring distinct identification procedures. More specifically, what is desired is a multi-dimensional separation method that provides for more accurate correlation of peaks across concentration distributions. Preferably, the method should work with a wide range of separation techniques.